Research on devR regulated cytotoxicity of Mycobacterium tuberculosis

doi:10.3969/j.issn.1673-8640.2015.06.016

Abstract

Abstract: Objective

To identify the influence of devR expression on the growth and cytotoxicity of tuberculosis.

Methods

Culture wild Mycobacterium bovis Bacillus Calmette-Guérin (BCG), devR knockout BCG (ΔdevR) and devR compensated BCG (devR.com) were identified for devR sequence, and the growth curves at different times were drawn under absorbance (A) 600 nm. A549 cells and Raw264.7 cells were infected with the strains at multiplicity of infection (MOI)=10. The apoptosis ratio was detected by flow cytometry, and lactate dehydrogenase (LDH) released by host cells was measured by enzyme-linked immunosorbent assay(ELISA) at 0, 24, 48 and 72 h. The expressions of apoptosis related genes, bcl-2 and bad, were determined by real-time quantitation polymerase chain reaction (PCR).

Results

Comparing to BCG and devR.com, the growth of ΔdevR was significantly accelerated at early phase (2-10 d, P<0.01). Apoptosis and necrosis ratio of A549 cells and Raw264.7 cells infected by ΔdevR were significantly higher than those infected by BCG and devR.com(P<0.05). All 3 strains could up-regulate the expressions of bcl-2 of A549 cells, and ΔdevR stimulated higher level of bcl-2 expression than BCG and devR.com. They also could increase the bad expression of Raw264.7 cells. ΔdevR induced more bad gene expression than BCG and devR.com.

Conclusions

The knockout of devR could enhance the cytotoxicity of BCG.

Key words: devR, Bacillus Calmette-Guérin, Infection, Apoptosis, Lactate dehydrogenase

CLC Number:

R446.7

DANZHENG Jiancuo, MA Yueyun, ZHOU Lei, FU Xiaorui, SU Mingquan, HAO Xiaoke. Research on devR regulated cytotoxicity of Mycobacterium tuberculosis[J]. Laboratory Medicine, 2015, 30(6): 607-612.

Figures/Tables 5

References 19

[1]	World Health Organization.Global tuberculosis report 2013[R].Geneva:WHO, 2013.
[2]	SOARES SC, TROST E, RAMOS RT, et al.Genome sequence of Corynebacterium pseudotuberculosis biovar equi strain 258 and prediction of antigenic targets to improve biotechnological vaccine production[J].J Biotechnol, 2013, 167(2):135-141.
[3]	MACHADO D, PERDIGÃO J, RAMOS J, et al. High-level resistance to isoniazid and ethionamide in multidrug-resistant Mycobacterium tuberculosis of the Lisboa family is associated with inhA double mutations[J].J Antimicrob Chemother, 2013, 68(8):1728-1732.
[4]	ZHANG L, ZHANG H, ZHAO Y, et al.Effects of Mycobacterium tuberculosis ESAT-6/CFP-10 fusion protein on the autophagy function of mouse macrophages[J].DNA Cell Biol,2012,31(2):171-179.
[5]	DESVIGNES L, WOLF AJ, ERNST JD.Dynamic roles of type Ⅰ and type Ⅱ IFNs in early infection with Mycobacterium tuberculosis[J].J Immunol,2012,188(12):6205-6215.
[6]	SICA A, MANTOVANI A.Macrophage plasticity and polarization: in vivo veritas[J].J Clin Invest,2012,122(3):787-795.
[7]	DEVASUNDARAM S, DEENADAYALAN A, RAJA A.In silico analysis of potential human T cell antigens from Mycobacterium tuberculosis for the development of subunit vaccines against tuberculosis[J].Immunol Invest,2014,43(2):137-159.
[8]	BE NA, BISHAI WR, JAIN SK.Role of Mycobacterium tuberculosis pknD in the pathogenesis of central nervous system tuberculosis[J].BMC Microbiol,2012,12:7.
[9]	WAGNER D, MASER J, LAI B, et al.Elemental analysis of Mycobacterium avium-, Mycobacterium tuberculosis-, and Mycobacterium smegmatis-containing phagosomes indicates pathogen-induced microenvironments within the host cell's endosomal system[J].J Immunol, 2005, 174(3):1491-1500.
[10]	KAUR K, TANEJA NK, DHINGRA S, et al.DevR (DosR) mimetic peptides impair transcriptional regulation and survival of Mycobacterium tuberculosis under hypoxia by inhibiting the autokinase activity of DevS sensor kinase[J].BMC Microbiol,2014,14:195.
[11]	GAUTAM US, SIKRI K, VASHIST A, et al.Essentiality of DevR/DosR interaction with SigA for the dormancy survival program in Mycobacterium tuberculosis[J].J Bacteriol,2014,196(4):790-799.
[12]	BEIER D, GROSS R.Regulation of bacterial virulence by two-component systems[J].Curr Opin Microbiol, 2006, 9(2):143-152.
[13]	GENGENBACHER M, KAUFMANN SH.Mycobacterium tuberculosis: success through dormancy[J].FEMS Microbiol Rev,2012,36(3):514-532.
[14]	BETTS JC, LUKEY PT, ROBB LC, et al.Evaluation of a nutrient starvation model of Mycobacterium tuberculosis persistence by gene and protein expression profiling[J].Mol Microbiol, 2002, 43(3):717-731.
[15]	VOSKUIL MI, SCHNAPPINGER D, VISCONTI KC, et al.Inhibition of respiration by nitric oxide induces a Mycobacterium tuberculosis dormancy program[J].J Exp Med, 2003, 198(5):705-713.
[16]	SHILOH MU, MANZANILLO P, COX JS.Mycobacterium tuberculosis senses host-derived carbon monoxide during macrophage infection[J].Cell Host Microbe, 2008, 3(5):323-330.
[17]	TANEJA NK, DHINGRA S, MITTAL A, et al.Mycobacterium tuberculosis transcriptional adaptation, growth arrest and dormancy phenotype development is triggered by vitamin C[J].PLoS One, 2010, 5(5):e10860.
[18]	SELVARAJ S, SAMBANDAM V, SARDAR D, et al.In silico analysis of DosR regulon proteins of Mycobacterium tuberculosis[J].Gene,2012,506(1):233-241.
[19]	HU Y, MOVAHEDZADEH F, STOKER NG, et al.Deletion of the Mycobacterium tuberculosis alpha-crystallin-like hspX gene causes increased bacterial growth in vivo[J].Infect Immun, 2006, 74(2):861-868.